The HIV-1 epidemic has resulted in approximately 5 million new infections in 2004 for a total of 40 million people living with HIV/AIDS. A preventative vaccine for HIV-1 is urgently needed. It is generally agreed that an antibody-inducing vaccine will be essential for an efficacious public health strategy. In previous work, we have identified several novel immunogens based on the HIV-1 envelope protein that demonstrate an improved ability to induce neutralizing antibodies to primary isolates of the virus. Each of these novel immunogens is heavily glycosylated, which is typical of the HIV-1 envelope. Previous studies of envelope glycosylation suggest the hypothesis that modifications of glycosylation can lead to further improvements in the immunogenicity of envelope proteins. Beginning with three lead immunogens, we will systematically evaluate the effect of glycosylation on the ability of these proteins to induce broadly cross-reactive antibodies to clinically relevant isolates of HIV-1. In the present Phase I SBIR Proposal, a large number of combinatorial glycosylation variants will be created using a novel in vitro gene recombination technology. The biosynthesis of the envelope variants as well as their ability to bind CD4 and various neutralizing monoclonal antibodies will be evaluated. The output of the current Phase I workplan will be a collection of glycosylation variants that are suitable for testing by immunization in animals. Only in vivo studies can truly measure the ability to induce virus-neutralizing antibodies. This latter work will be the subject of a Phase II SBIR application. The ultimate goal is to identify one or a small number of immunogens that can be tested for immunogenicity in clinical trials in humans. The Specific Aims of the current Phase I SBIR submission are: (1) create libraries of glycosylation variants using in vitro recombination; (2) isolate thousands of individual clones from the libraries; and (3) select variants that are well secreted, properly folded, and carry the known conserved neutralizing epitopes. This Proposal is targeted to one of the Areas of Interest of the Division of AIDS, NIAID relative to the preclinical development and evaluation of HIV vaccines, adjuvants, delivery systems, and new formulations for agents to prevent HIV infection.